Field
This invention pertains to devices, apparatus, and methods for potentiation of agglutination reactions and more generally to antigen:antibody reactions in microfluidic devices. Antigen:antibody reactions involving agglutination are useful in blood typing, in crossmatching for blood transfusion, and in immunodiagnostic agglutination assays in general.
Description of Related Art
Administration of blood in the form of packed erythrocytes or whole blood is often useful in the treatment of trauma, hypovolemic shock, anemia and clotting disorders, and generally requires, at a minimum, characterization of the donor blood so as to match the ABO blood type of the donor and recipient, and more generally requires a crossmatch. This is done to avoid a hemolytic transfusion reaction in which red cells having a major incompatibility antigen are inadvertently administered to a recipient having an antibody to that antigen, and also to avoid the minor side reaction in which a red cell antigen in the recipient's blood is attacked by antibodies in the plasma of the donor. Serious consequences such as kidney failure or splenic rupture can result from a transfusion of mismatched blood.
Because blood, once collected from a donor, has a limited shelf life, it is desirable to collect blood from donors of the blood types most required for transfusion. Blood not used promptly is generally discarded or processed for bulk protein, a less valuable use. However, in current practice, the donor's blood type and the actual need for that donor's blood type is not determined until after the “unit” of donor blood has been collected, by which time, unfortunately, it is often determined that available units of the donor blood type are already available in excess. Therefore, pre-donation screening of donors for ABO blood type could enhance blood bank operations by identifying donors with the required blood types prior to donation; and conversely by avoiding unnecessary blood collection, reducing overall time and cost.
Forward ABO typing is thus a valuable tool for donor screening. This practice further permits an immediate decision as to which donors are to be recruited into an apheresis program to specifically provide plasma or to provide platelets of a needed blood type and thereby reduce the wastage of a valuable resource and effort.
A more complete screen prior to transfusion requires that both donor and recipient also be screened for Rhesus (Rh) blood group antigen compatibility, particularly D antigen. The ABO antigens are glycolipids while Rh antigens are proteins. Although anti-D antibody is relatively uncommon, it has been shown that administration of D-incompatible blood frequently results in formation of antibodies in the recipient that can later cause a major hemolytic reaction upon subsequent transfusion and can also cause injury to a fetus in utero. Therefore, forward D screening is also essential for optimal use of blood bank resources. D antigen screening has been problematic due to the “weak” or “incomplete” characteristics of the antigen or antibody.
Most blood centers also test for the C and E antigens of the Rhesus group because only donations negative for C, D, and E may be truly labelled Rh negative. A pooled antiserum with reactivity against all members of the Rhesus group is available for this purpose.
Less common antibodies are also sometimes responsible for transfusion reactions, and can be screened for with a Coombs' test [Coombs, RRA. 1945. A new test for the detection of weak and ‘incomplete’ Rh agglutinins. 26:255-66], using anti-human globulin (AHG) reagent and also by the agglutination potentiation method disclosed in European Patent EP0039195B1 and by related chemical potentiation methods.
Also potentially problematic are atypical results due to variants in the ABO blood antigens, for example the need to distinguish “weak A” donors and to identify A subtypes A1 and A2.
The availability of standardized monoclonal antisera has made possible rapid slide and tube agglutination tests for the major blood groups A, B and AB. With controlled heating and use of agglutination potentiation reagents such as 30% albumin, blood group D can also be tested manually by slide agglutination or tube tests. Means for controlled heating and centrifugation, however, are not always available where blood donors are being screened. Also, during the testing and disposal of the slides or tubes, technicians using tube and slide test methods risk contact with the blood samples. Overall, these manual blood typing methods are labor and/or time intensive, require liquid reagents with limited shelf life unless refrigerated, require skilled technicians, and require additional equipment or overhead to operate.
U.S. Pat. No. 2,770,572 to Eldon, issued in 1956, describes a stiff paper card faced with a layer of cellulosic resin or gelatin on which are dried antisera to A, B and Rh antigens in premeasured amounts. Dextran, gum acacia, polyvinylpyrrolidinone, high molecular weight polyalcohols, or albumin are used to potentiate Rh agglutination. The Eldon Home Kit (or Eldoncard), is sold today through a number of different distributors as well as via the internet. Use of the Eldoncard is somewhat cumbersome in that the user is required to perform four “precise” water transfers of one drop each, and four distinct blood transfers to the card. Eldon also instructs the user to “carefully respect the times prescribed for tilting (4×10 seconds per angle) the card.” Current time to result is reported to be two to five minutes per test. While once seen as beneficial because the cardstock with agglutinated blood can be dried and stored as a permanent record, open format tests such as this are now of concern because those who handle the cards are directly exposed to potentially infectious materials. Permanent records are better kept electronically.
Enclosed devices are taught in U.S. Pat. No. 4,756,884 to Hillman, which teaches a plastic capillary flow device for detecting antigens in blood samples. The devices are constructed of generally poorly hydrophilic plastics such as polyethylene terephthalate glycol (PETG), polyester (Mylar®), polyvinylchloride, polystyrene, polycarbonate, or styrene acrylonitrile, or acrylonitrile-butadiene-styrene (ABS). In order to improve blood flow and reagent coating, argon plasma etching or corona discharge are taught (Col 14; lines 44-65).
The Hillman devices are generally constructed of layers and are adhered to seal around the edges of the internal channels and chambers by ultrasonic welding. Alternatively, double-sided adhesive tape cut to fit around the channels and chambers is sandwiched between the layers (Col 15, lines 5-57). One or more antibody reagents, combined with dissolution agents such as surfactants, polyols, sugars and the like, are supplied in the reaction chamber through which the blood sample flows. As further taught in U.S. Pat. No. 5,140,161, also to Hillman, these methods detect agglutination by reliance on stoppage of flow as the result of a plugged channel (Col 11; lines 1-13).
EP 0456699 to Vale describes a similar enclosed plastic device in which reagents for red cell agglutination are impregnated in permeable membranes disposed in reaction chambers and will dissolve when whole blood flows through the reaction chambers. Agglutination is detected by evidence of blockage of flow downstream from the reaction chambers.
U.S. Pat. No. 6,488,896 to Klein, which is co-owned by the present Applicant, teaches a device for blood typing which sought to overcome the above limitations by combining the blood sample with a liquid antiserum in a plastic device under conditions where the two streams did not mix by turbulence but instead flowed contiguously in parallel along a serpentine path under gravity, during which red cells sedimented from the upper blood layer into the lower antiserum layer, where agglutination takes place. In order to achieve this effect, liquid reagent and blood were first dispensed into two chambers which were connected to a common serpentine channel. The device was then tilted on end to commence flow and a positive agglutination reaction was evidenced by the gross visual appearance of red cell aggregates in the serpentine channel or by blockage of flow in an optional filter member at the end of the channel. The disadvantages are that the blood sample can be tested for only one antigen at a time in this device, that dry reagents cannot be used in the device, and that rivulets tend to form under gravitational flow instead of the liquid smoothly filling the reaction chamber. Also owned by the Applicant is U.S. Pat. No. 6,743,399, which proposed detection of red cell agglutination by blockage of flow (Col 8-9; lines 63-4).
WO 2004/065930 to Saltsman, which is co-owned by the present Applicant, describes devices for blood typing with three reaction channels for detecting A, B, O and D blood groups simultaneously (FIG. 6) using liquid reagents. Multiple optical viewing areas over the reaction channels allowed the user to type the blood sample by visually detecting agglutinated red cells in a sealed disposable unit. An in-line liquid impermeable barrier was used to prevent escape of the blood sample from the device. Suction pressure generated by a diaphragm pump downstream from the reaction channel was used to draw the blood sample and reagent liquid through the device and into a waste chamber. Thus the device could be operated while flat on a bench and did not require gravity-assisted flow.
WO 2006/009724 to Saltsman, which is also co-owned by the present Applicant, describes devices for blood typing with three channels for detecting A, B, O and D blood groups simultaneously (see FIG. 6 of WO2006/009724), using dried reagents placed in the device during manufacture. The dried reagents were first rehydrated before admixture with the blood sample. These devices allowed only for forward blood grouping and not for crossmatching and have not been used for any testing except A, B and D blood agglutinins. Suction pressure generated by a diaphragm pump downstream from the reaction channel was used to draw the blood sample and reagent liquid through the device and into a waste chamber. D antigen testing was found to be generally unreliable in these devices.
Gel tests (Biovue, Diamed, BioRad and Akers) use gel chromatography to detect agglutination. See for example U.S. Pat. No. 5,552,064 to Chachowiski, U.S. Pat. No. 5,830,411 to Gisper-Sauch, U.S. Pat. No. 5,338,689 to Lapierre, U.S. Pat. No. 5,905,028 to Frame, and U.S. Pat. No. 6,114,179 to Lapierre. See also Lapierre et al (1990. The gel test: A new way to detect red cell antigen-antibody reactions. Transfusion 30:109). In general, such products are complex to manufacture, require substantial time and effort to load reagents, process the test, and generally allow a certain percentage of errors when compared to reference methods. (See for example Migeot, V et al. 2002. Reliability of bedside ABO testing before transfusion. Transfusion 42:1348-55; Dujardin P P et al. 2000. Errors in interpreting the pretransfusion bedside compatibility test. Vox Sang 78:37-43; Ingrand P et al. Reliability of the pre-transfusion bedside compatibility test. Transfusion 38:1030-36.) In one such study, a bedside error rate of 30-35% of samples was obtained when trained nurses administered the test. The Vu-Test®, marketed by Medigis and Baxter failed commercially and was withdrawn from market in France following poor performance.
These tests also require a gel reader and a centrifuge. Some type of physical barrier (e.g., column/gel matrices, lateral flow strips, or separation membranes) is used separate free red cells and agglutinated red cell clumps. The separation step inherently results in a longer time to test result.
Also in development is the MDmulticard (Medion Diagnostics, Germany). The MDmulticard as described in WO 2005/090970 and US 2007/0248983 is essentially a lateral flow strip method. The test requires two extra sample preparation steps: a red cell wash step and a pre-dilution step, both of which slow testing and require skilled technicians. Following preparation of the dilute washed cell suspension, results reportedly are obtained in approximately 5 minutes. As such, the test does not meet the need for more immediate test results. Reagent red cells having a 5 week shelf-life, with refrigeration, are available for reverse typing in this test package.
LISS (Low Ionic Strength Saline), PEG, albumin or other conglutinins are used to promote sensitivity in test packages that use washed red cells, increasing the complexity of the product kit and requiring specialized conditions for reagent storage and liquid handling tools such as pipets which require calibration. Another lateral flow test is described in U.S. Pat. Nos. 5,231,035 and 5,565,366, and has been commercialized by Akers Biosciences.
Unlike systems by Medion Diagnostics, Biovue, Diamed, BioRad, Akers and others which rely on washed red cell suspensions, the methods of the current invention use whole blood for the testing, speeding time to completed test and permitting direct testing from capillary tubes drawn by fingerstick, for example.
This field has had its commercial failures and has proved unpredictable. There is a need in the art for improvements that overcome the above disadvantages and reliably potentiate antigen-antibody reactions, particularly agglutinations involving “incomplete” antibodies or weak agglutination. Such improvements are broadly applicable in a variety of immunodiagnostic applications.
A preferred solution to the problem of forward blood typing will use whole blood, eliminating the need for washed cells. In view of the need for rapidly screening potential donors, testing requiring longer than two minutes is problematic. Testing should not require special incubation conditions and testing apparatus. There is a need for a two minute test that is self-contained so that it can be performed without special equipment or sample preparation.
Similar devices also may find application in crossmatching of blood donors and recipients prior to transfusion. Other applications are found more generally in agglutination-based immunoassays, such as direct and indirect Coombs testing and in testing for microbial serotypes or humoral responses to infections, for example.